First report of kdr mutations in the voltage-gated sodium channel gene in the arbovirus vector, Aedes aegypti, from Nouakchott, Mauritania

Background Since 2014, dengue epidemics have occurred almost annually in Nouakchott, the capital city of Mauritania, coinciding with the recent establishment of Aedes aegypti, the primary vector of dengue, in the city. Anopheles arabiensis, the primary vector of malaria, is also abundant not only in Nouakchott but also in most areas of the country. Resistance to insecticides has been studied in An. arabiensis but not in Ae. aegypti in Mauritania. The objective of the present study was to establish the baseline data on the frequencies of knockdown resistance (kdr) mutations in the voltage-gated sodium channel (vgsc) gene in Ae. aegypti collected in Nouakchott to improve vector control. Methods Resting Ae. aegypti mosquitoes were collected in 2017 and 2018 in Teyarett and Dar Naim districts in Nouakchott using a battery-powered aspirator. Polymerase chain reaction (PCR) and DNA sequencing were performed to detect the presence of five kdr mutations known to be associated with pyrethroid resistance: L982W, S989P, I1011M/G, V1016G/I, and F1534C. Results A total of 100 female Ae. aegypti mosquitoes were identified among collected resting culicid fauna, of which 60% (60/100) were unfed, 12% (12/100) freshly blood-fed, and 28% (28/100) gravid. Among the mutations investigated in this study, 989P, 1016G, and 1534C were found to be widespread, with the frequencies of 0.43, 0.44, and 0.55, respectively. Mutations were not found in codons 982 and 1011. No other mutations were detected within the fragments analyzed in this study. Genotype distribution did not deviate from Hardy–Weinberg equilibrium. The most frequent co-occurring point mutation patterns among Ae. aegypti mosquitoes were the heterozygous individuals 989SP/1016VG/1534FC detected in 45.1% of mosquitoes. In addition, homozygous mutant 1534CC co-occurred simultaneously with homozygous wild type 989SS and 1016VV in 30.5% of mosquito specimens. Inversely, homozygous wild-type 1534FF co-occurred simultaneously with homozygous mutant 989PP and 1016GG in 19.5% of the mosquitoes. Conclusions To our knowledge, this is the first study reporting the presence of three point mutations in the vgsc gene of Ae. aegypti in Mauritania. The findings of the present study are alarming because they predict a high level of resistance to pyrethroid insecticides which are commonly used in vector control in the country. Therefore, further studies are urgently needed, in particular phenotypic characterization of insecticide resistance using the standardized test. Graphical Abstract


Introduction
Dengue is the most widespread mosquito-borne viral disease in the world [1].In Mauritania, dengue has also been a significant public health problem since the first documented dengue outbreak in 2014 in Nouakchott, the capital city [2,3].The emergence of dengue coincided with the first detection of Aedes aegypti in the city [4].Since 2014, dengue cases and outbreaks have been reported annually in Nouakchott and other urban settings, particularly in the Oasian city of Atar and the mining city of Zouérat in the northern Saharan zone [5][6][7].
Aedes aegypti is the major vector of dengue as well as three other arboviruses of public health importance, namely yellow fever, chikungunya, and Zika [8].This mosquito species is well adapted to thrive in urban environments where suitable human-made habitats for mosquito vectors exist and contact between mosquito vectors and human hosts is frequent, facilitating arbovirus transmission [9].At present, there is no specific treatment for dengue.Only one licensed vaccine (Dengvaxia ® ) is currently available in several Asian and Latin American countries, but it is not recommended for the general population; it may be administered only in children (aged 9-16 years) with a previous history of dengue residing in endemic areas [10].Vector control based on the reduction of larval habitats and space-spraying of insecticides remains the most common approach to reduce the incidence of dengue in the general population [11,12].
Pyrethroids are insecticides most commonly used to control adult Ae. aegypti [13].Knockdown resistance (kdr) is one of the most important mechanisms of resistance to pesticides documented globally in a large number of arthropods of medical importance [14].Pyrethroids (e.g., deltamethrin, permethrin) and organochlorines (e.g., dichlorodiphenyltrichloroethane, DDT) are insecticides that trigger the kdr phenotype [15].Mutations in the gene encoding voltage-gated sodium channels (VGSC) are associated with the kdr phenotype.Overall, 12 non-synonymous mutations varying in frequency, geographical distribution, and impact on resistance phenotype, have so far been detected across the world, of which six (V410L, L982W + F1534C, S989P, I1011M/V, V1016G/I, and F1534C) have been demonstrated to be associated with pyrethroid resistance in Ae. aegypti [13,16].The mutations S989P, I1011M or V, V1016G or I, and F1534C occur within domains II and III of VGSC.Recent studies have shown that three of these mutations, 1016G, 1016I, and 1534C, are widely distributed and detected in pyrethroid-resistant populations of Aedes mosquitoes in several African countries, including Cameroon, Côte d'Ivoire, Burkina Faso, Cape Verde, and Ghana [17][18][19][20][21][22].
Historically, the first vector control program targeting malaria vector in Mauritania was implemented in the early 1990s.It was first based on the promotion of the use of insecticide-treated nets (ITNs), and then a few years later, long-lasting insecticide-treated nets (LLINs) were introduced to replace the conventional ITNs.Mass distribution of pyrethroid-impregnated (deltamethrin and permethrin) mosquito nets has been the main vector control intervention since 1997.Indoor residual spraying and larval control are also part of the priorities recently identified in the national malaria control policy, but they have not yet received the necessary funding for their implementation.Studies assessing the impact of LLINs in reducing the incidence of malaria and other vector-borne diseases in the country are lacking.
Among mosquitoes of medical importance, the phenotypic and molecular basis of resistance to insecticides has been studied in the malaria vector Anopheles arabiensis but not in Ae. aegypti in Mauritania [23,24].The present study assessed the frequencies of six point mutations known to confer pyrethroid resistance in Ae. aegypti collected in Nouakchott in 2017-2018, i.e., 3 years after the first documented dengue outbreak following the detection of the arboviral vector in the city [2,4], with the objective of establishing baseline data for future arbovirus surveillance and vector control strategies.

Study sites
The study was part of the entomological surveillance of malaria and dengue vectors in Nouakchott, the capital city of Mauritania.It was carried out in the districts of Dar Naim and Teyarett located in northern and northeastern areas of Nouakchott.A detailed description of the study sites was presented in our earlier works [4,23,25].In Teyarett and Dar Naim, malaria is known to be endemic, and several epidemiological, parasitological, and entomological studies on malaria in Nouakchott have been conducted in these two districts [25][26][27][28].Teyarett was also the epicenter of the first dengue outbreak that occurred in Mauritania in 2014.Dengue cases have been reported from the district of Dar Naim as well.Based on clinical reports of dengue fever occurring in Nouakchott, entomological monitoring of Ae. aegypti, the most probable vector of dengue, was initiated in these two districts 3 years (i.e., from 2017) after the first detection of this mosquito species in Nouakchott.

Mosquito sampling
A battery-powered Prokopack aspirator (John W. Hock Company, Gainsville, FL, USA) was used to collect indoor resting culicid fauna.The collection was carried out during the morning (between 07:00 and 09:00) for one to four consecutive days per month in each site (five houses/site and two rooms per house).Overall, mosquitoes were collected from March to June 2017 and during the month of September 2018.Upon collection, mosquitoes were euthanized by placing them in a −20 °C freezer.They were then sorted by species and sex using appropriate taxonomic keys [29].Female Ae. aegypti were further classified according to abdominal status (unfed, fed, semi-gravid, or gravid), then stored at −80 °C until use for kdr genotyping.

Genotyping kdr
Aedes aegypti specimens were placed in microtubes with 5-mm diameter stainless steel beads for use with the Tis-sueLyser system (Qiagen S.A.S., Courtaboeuf, France) and 205 µl of lysis buffer (25 µl of proteinase K and 180 µl of T1 buffer in the NucleoSpin ® 96 tissue core kit).Specimens were crushed in the TissueLyser II (Qiagen S.A.S., Courtaboeuf, France) with three cycles of 1 min at a frequency of 30/s separated by 20 s of pause time.Samples were incubated at 70 °C for 1 h.Mosquito DNA was extracted using the NucleoSpin ® 96 tissue core kit (Macherey Nagel AG, Oensingen, Switzerland) following the manufacturer's instructions.
Purification of PCR products and sequencing were outsourced to Biofidal Themis (Vaulx-en-Velin, France).Each fragment was sequenced from both 5′ and 3′ ends.The sequences were aligned and compared with those of GenBank accession nos.MK977832.1 and MF794973.1 using Molecular Evolutionary Genetics Analysis (MEGA, version 10) software.

Statistical analysis
Data were entered into an Excel spreadsheet (Microsoft Office Excel 2007, Microsoft Corporation, Redmond, WA, USA).The proportions of unfed, fed, and gravid resting females were compared using Fisher's exact test.Hardy-Weinberg equilibrium for observed genotype frequencies for each kdr mutation was calculated using the Chi-square test.Statistical significance was set at P < 0.05.All data were analyzed using Excel software or MedCalc ® statistical software version 20.115 (MedCalc Software Ltd, Ostend, Belgium; https:// www.medca lc.org; 2022).

Results
A total of 100 Ae. aegypti female adults were collected in 2017 (n = 50) and 2018 (n = 50) during the entomological surveillance of Anopheles and Aedes mosquitoes in Teyarett and Dar Naim districts of Nouakchott.

Discussion
The present study was conducted to determine the frequencies of kdr mutations in adult female Ae.aegypti collected a few years after the establishment of the arbovirus vector in Nouakchott.The collection of indoor resting female mosquitoes showed that a high proportion of Aedes mosquitoes were unfed.Studies conducted in Asia and Latin America showed that Ae. aegypti populations are typically endophagic and endophilic, resting and feeding in human habitations [30,31]; in West Africa, however, studies revealed a broader range of resting behaviors [32][33][34].For instance, studies conducted in Niger showed endophilic and endophagic behavior of Ae. aegypti populations [32], while in Ghana and Senegal, Ae. aegypti populations were collected mostly outdoors rather than indoors [33,34].In the present study, Ae. aegypti exhibited a similar behavior as that reported from Senegal and Ghana, but given the low number of unfed mosquitoes, a solid conclusion cannot be drawn at present.However, this finding is important for vector control strategies and points to the need for more in-depth studies on the bioecology of Ae. aegypti in Mauritania.
Furthermore, we analyzed the prevalence of kdr mutations in the vgsc gene of individual Ae.aegypti mosquitoes in Nouakchott.Two major African-type kdr mutations strongly linked to pyrethroid resistance, 1016G and 1534C, were found in Ae. aegypti analyzed in this study with high allelic frequencies: 1016G (0.44) and 1534C (0.55).For the first time, the Asian-type kdr mutation linked to pyrethroid resistance 989P [35] was found in Mauritanian Ae. aegypti with high allelic frequency (0.43).More interestingly, a high proportion (45.1%) of Aedes mosquitoes harbored the triple mutations 989SP/1016VG/1534FC with the heterozygous status, suggesting a potentially high level of resistance to pyrethroids.Similar findings were reported from Myanmar [36].However, in a study conducted in Senegal [37], no mutations in the vgsc gene associated with pyrethroid resistance were found despite the phenotypic resistance to pyrethroids observed in Ae. aegypti populations.In that study, insecticide resistance of Ae. aegypti population was found to be associated with a remarkably high (20-to 70-fold) overexpression of the major detoxification genes, suggesting that insecticide resistance in investigated Ae. aegypti specimens was likely to be mediated by metabolic mechanisms.In another study conducted in Cape Verde [38], many of the mutations commonly associated with insecticide resistance, including V1016G/I, S989P, and F1534C, were detected in Ae. aegypti screened samples, but insecticide resistance/susceptibility phenotype was not determined in that study.However, in two West African countries, Ghana and Burkina Faso, two of the mutations detected in the present study (F1534C and V1016G) were found at a high frequency among pyrethroid-resistant Ae. aegypti populations [19,20,39,40].These findings on high frequencies of kdr mutations are not surprising since pyrethroids remain the most commonly used class of insecticides in vector control worldwide [41].
Studies on kdr mutations in Ae. aegypti in West Africa are still scarce.However, point mutations reported here, in particular F1534C and V1016G, have been shown to play an important role in conferring insecticide resistance to local Aedes mosquitoes.Indeed, since its first detection in Ae. aegypti populations from Ghana [42], Aedes mosquitoes with the F1534C mutation have been spreading in Africa.This mutation has since then been reported from Côte d'Ivoire [18], Cameroon [43], Angola, and Cape Verde [20].In Burkina Faso, the F1534C substitution was found to be almost fixed in the populations of Ae. aegypti in the capital city, Ouagadougou [39].Concomitant presence of these point mutations was also reported to strongly increase the level of pyrethroid resistance in individual mosquitoes [44].In our study, high proportions of female Aedes mosquitoes displayed double or triple mutations, possibly implying a high level of pyrethroid resistance [45].
In our study, we observed that homozygous wild-type genotypes at codon 1534 co-occurred with homozygous mutants at 989P and 1016G, and vice versa.This finding makes it more important to look for the other mutations present in domains II and III of VGSC in Aedes.In Asia, but not yet in Africa, novel mutations have been reported to be associated with pyrethroid resistance: A1007G [46], T1520I [47], and F1534L [48].In our study, we did not observe F1534L and A1007G.Future studies in Africa should include monitoring of these mutations.
In Mauritania, the first report of Ae. aegypti in Nouakchott dates back to 2014 [4].However, this situation alone cannot explain the potentially high frequencies of kdr mutations observed among Ae.aegypti mosquitoes collected 3 years later in two districts of Nouakchott.Therefore, it can be hypothesized that Aedes mosquitoes harboring different kdr point mutations may have been introduced in Nouakchott from other sites or countries, followed by cross-mating between them, resulting in highly heterogeneous mosquito populations.However, the unknown impact and contribution of vector control campaigns through aerial spraying of insecticide carried out in recent years to reduce mosquito nuisance, particularly in Nouakchott, cannot be overlooked [28].
With the rapid and disorganized urbanization process that Nouakchott has been experiencing over the past 20 years, along with the rapidly growing human population and massive importation of second-hand tires resulting from the growing demand of used cars imported from Europe, Nouakchott offers an environment that is conducive to maintaining the habitats of Ae. aegypti.Indeed, studies from Kenya and Tanzania showed that tires provide good breeding sites for Aedes mosquitoes and are responsible for producing > 30% of immatures collected from larval habitats in urban areas [49,50].The alarming findings of the present study call for an effective vector control strategy based on reinforced entomological surveillance, integrated approaches to control both larval and adult stages of Aedes mosquitoes, and fully engaged community-led actions.
The main limitations of the present study are that the insecticide resistance/susceptibility phenotype of genotyped mosquitoes was not assessed during the study to correlate the observed genotypes with their corresponding insecticide resistance phenotypes and that the recently detected V410L kdr mutation associated with high levels of resistance to both types I and II of pyrethroids was not searched [51].Another limitation was that our sample size was small.

Conclusions
This is the first report on the presence of kdr mutations in Ae. aegypti from Mauritania since this mosquito species was established in Nouakchott.The findings of the present study are alarming because they predict a high level of resistance to pyrethroids in the Ae.aegypti population found in the capital city.Further studies are urgently needed, in particular phenotypic characterization of insecticide resistance using the standardized test in parallel with genotyping more recent collection of Ae. aegypti mosquitoes.Furthermore, as insecticide resistance negatively impacts vector control, regular surveillance of the resistance/susceptibility status of the mosquito vector population should be implemented, with a primary focus on the chemical insecticides used in vector control in Mauritania.

Table 1
Abdominal status of resting female Aedes aegypti mosquitoes collected in Nouakchott, Mauritania Comparison of frequencies between unfed and gravid female Ae.aegypti mosquitoes a Including half-gravid female b

Table 2
presents the results of vgsc genotyping.All successfully genotyped mosquitoes were homozygous wildtype (i.e., monomorphic) for the kdr codons 982 (n = 81) and 1011 (n = 85).The three remaining kdr point mutations, 989P, 1016G, and 1534C, were found among the investigated Aedes mosquitoes with allelic frequencies of 0.43, 0.44, and 0.55, respectively.There were no statistical differences in the frequencies of kdr point mutations in Aedes mosquitoes collected in 2017 compared with those collected in 2018 (P = 0.55, Fisher's exact test).

Table 2
Allelic and genotypic frequencies of kdr mutations in female Aedes aegypti mosquitoes, Nouakchott, Mauritania S, susceptible; R, resistant; UI, uninterpretable; kdr, knockdown resistance; HWE, Hardy-Weinberg equilibrium at 5% significance level; NA, not applicable a Frequency of mutant allele